Marakhonov et al. BMC Medical Genetics 2020, 21(Suppl 1):156
https://doi.org/10.1186/s12881-020-01093-z

CASE REPORTOpen Access

Prenatal diagnosis of Norrie disease after
whole exome sequencing of an affected
proband during an ongoing pregnancy: acase report
Andrey V. Marakhonov1* , Irina A. Mishina1, Vitaly V. Kadyshev1, Svetlana A. Repina1, Maria F. Shurygina2,
Olga A. Shchagina1, Natalya N. Vasserman1, Tatyana A. Vasilyeva1, Sergey I. Kutsev1 and Rena A. Zinchenko1,3
From 11th International Young Scientists School “Systems Biology and Bioinformatics” – SBB-2019

Abstract
Background: Hereditary ophthalmic pathology is a genetically heterogeneous group of diseases that occur either as anisolated eye disorder or as a symptom of hereditary syndromes (chromosomal or monogenic). Thus, a diagnostic searchin some cases of ophthalmic pathology can be time- and cost-consuming. The most challenging situation can arise whenprenatal diagnosis is needed during an ongoing pregnancy.
Case presentation: A family was referred to the Research Centre for Medical Genetics (RCMG) for childbirth risk prognosis at
7–8 week of gestation because a previous child, a six-year-old boy, has congenital aniridia, glaucoma, retinal detachment,
severe psychomotor delay, and lack of speech and has had several ophthalmic surgeries. The affected child had beenpreviously tested for PAX6 mutations and 11p13 copy number variations, which revealed no changes. Considering the lack ofpathogenic changes and precise diagnosis for the affected boy, NGS sequencing of clinically relevant genes was performed
for the ongoing pregnancy; it revealed a novel hemizygous substitution NM_000266.3(NDP):c.385G > T, p.(Glu129*), in theNDP gene, which is associated with Norrie disease (OMIM #310600). Subsequent Sanger validation of the affected boy and hismother confirmed the identified substitution inherited in X-linked recessive mode. Amniotic fluid testing revealed the fetus
was hemizygous for the variant and lead to the decision of the family to interrupt the pregnancy. Complications whichdeveloped during the termination of pregnancy required hysterectomy due to medical necessity.
(Continued on next page)* Correspondence: marakhonov@generesearch.ru1
Laboratory of Genetic Epidemiology, Research Centre for Medical Genetics,
Moscow, Russian Federation
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(Continued from previous page)

Conclusions: Clinical polymorphism of hereditary ophthalmic pathology can severely complicate establishment of an exact
diagnosis and make it time- and cost-consuming. NGS appears to be the method-of-choice in complicated cases, and this
could substantially hasten the establishment of a diagnosis and genetic risk estimation.
Keywords: Hereditary eye pathology, Clinical heterogeneity, NGS, NDP, Norrie diseaseBackground
Hereditary eye pathology is a genetically heterogeneousgroup of diseases that occur either as an isolated ocular disorder or as a symptom of syndromes (both chromosomaland monogenic). We present here the results of DNA diagnosis in a patient with complex ocular phenotype associatedwith severe systemic involvement using next-generation sequencing (NGS), not only to perform differential diagnosis
but also to determine if such a complex clinical picture hasa single etiology or is a result of an interaction of factorsthat could have both hereditary and environmental origin.
The case is much more complicated because the proband’s
mother had an ongoing pregnancy, thus the DNA diagnosis
was performed with urgent need for genetic risk estimation
as well as subsequent prenatal diagnosis.

Fig. 1 Pedigree of the family

Case presentation
A family referred to the Research Centre for Medical
Genetics (RCMG) for risk-affected childbirth prognosis
at 7–8 week of gestation because a previous child, a sixyear-old boy, has congenital glaucoma, bilateral retinal
detachment, and severe psychomotor delay and has had
several ophthalmic surgeries (Fig. 1).
For the first time the proband (a full-term newborn)
was examined by an ophthalmologist at the age of 4months. Clinical presentation included roving eye movements and abnormal gaze behavior, but he had positive
light perception and toy tracking. Intraocular pressure
was 21 and 17 mmHg (OD and OS, respectively). B-scanultrasonography revealed a total funnel retinal detachment, iris aplasia in both eyes, an anterior chamber ofMarakhonov et al. BMC Medical Genetics 2020, 21(Suppl 1):156

normal depth with an open angle, and undifferentiatedtrabecular meshwork, right eye lens opacity in all layers,bilateral vitreous fibrosis, and numerous fibrotic transocular folds behind the lenses. The proband was diagnosed as having the consequences of intrauterine uveitis,
total retinal detachment, and secondary decompensatedglaucoma. He later underwent several surgeries: 2 lensectomies, 4 vitrectomies, retinotomy, and subretinal
aqueous drainage. No genetic test was performed at thattime. The family never sought genetic counseling.
By the date when the family was referred for genetic
counseling, the boy was blind in both eyes, had total bilateral retinal detachment, bilateral iris aplasia, left eye
phthisis bulbi, pronounced delay in psycho-emotional
development, lack of speech, and behavioral disorder.
Considering the ongoing pregnancy and the lack of aconfirmed diagnosis, we performed NGS analysis of clinically relevant genes. Whole exome sequencing (WES)
was performed using an BGISEQ-500 instrument withaverage on-target coverage 146× with MGIEasy Exome
Capture V4 (BGI) for library preparation (GenomedLtd., Moscow). Bioinformatic analysis was performedusing an in-house software pipeline which included quality control of raw reads (FastQC tool v. 0.11.5) followed byread mapping to the hg19 human genome assembly (bwamem v. 0.7.1), sorting of the alignments, marking duplicates (Picard Toolkit v. 2.18.14). Base recalibration andvariant calling were performed with GATK3.8. Variant annotation was done using Annovar tool (v. 2018Apr16).
Further filtering was performed by functional consequences and population frequencies according to theACMG recommendations [1] as well as clinical relevancedetermined by Human Phenotype Ontology database [2].
The analysis lasted 42 days and revealed a novel hemizygous
substitution,
NM_000266.3(NDP):c.385G > T,p.(Glu129*), in the NDP gene, which was associated with
Norrie disease (OMIM #310600) inherited in X-linked recessive mode. Subsequent validation of the affected childand his mother by Sanger sequencing confirmed the identified substitution. The substitution was not registered incontrol cohorts in the Genome Aggregation Database(gnomAD). The identified novel NDP sequence variant
c.385G > T, p.(Glu129*), forms a premature termination
codon (PTC). Although the variant affects only the mostC-terminal portion of the protein (129 out of 133 aminoacid residues), it eliminates the ultimate cysteine-131
residue that is involved in formation of an intermoleculardisulfide bond [3]. This should lead to the disruption of avery rigid structure of Norrin homodimer. Nearby PTC
forming pathogenic variants have been described –p.Cys128* and p.Cys131* [4, 5]. It was previously shownthat disruption of the dimer by either cysteine-to-alaninemutations of intermolecular disulfide bonds results in aloss of Norrin function [6, 7]. Norrin is an atypical Wnt

Page 3 of 5ligand that can activate β-catenin signaling through its
specific binding to the Frizzled4 (Fz4) receptor [8] to function as a ligand–receptor pair that controls vascular development in the retina and inner ear [6]. Pathogenicvariants in the NDP gene are associated with a spectrumof conditions – from milder familial exudative vitreoretinopathy to the systemic Norrie disease [9].
The clinical exome sequencing in the proband motivated amniotic fluid testing in his pregnant mother atthe 17th week of gestation, which revealed after additional 5 days that the fetus was male and hemizygous
for the variant. The family decided to interrupt the pregnancy after the prenatal test at the 20th week of gestation. Complications in the form of profuse uterine
bleeding developed during the pregnancy termination
required hysterectomy due to medical necessity.

Discussion and conclusions
Congenital vitreoretinopathy with retinal detachment isa highly heterogeneous group of disorders. Patients withgenetically determined disorders such as familial exudative vitreoretinopathy, Norrie disease, or Coats disease,
as well as non-hereditary retinopathy of prematurity orinflammation of the retina could have similar ocular
manifestations [9]. Norrie disease is a rare X-linked recessive disorder of retinal blood vessel development. According to Orphanet database, ~ 400 cases have been
described to date [10]. They are characterized by congenital vitreoretinopathy, avascularity of the peripheral
retina and its secondary neovascularization, fibrosis,
traction and retinal detachment, and blindness, which inabout 25–50% of the cases, is accompanied by progressive mental retardation and/or sensorineural deafness[11, 12]. In addition, some patients have more complex
phenotypes, including growth failure and seizure. Radiological examination of some patients with Norrie disease
has demonstrated brain and cerebellar atrophy [13].
The cellular basis for Norrie disease is an insufficiency
of Frizzled signaling, and more specifically the Frizzled-4
ligand Norrin. Norrin functions as an activator of the canonical Wnt/β-catenin signaling pathway in capillary
endothelial cells in the retina and in other structures[12, 14]. Norrin is engaged in Müller glia in the retina,
in stria vascularis and a capillary plexus of cochlea, inforebrain and midbrain astrocytes, and in cerebellum
Bergman glia. Norrin is also required for blood–brain
barrier integrity and maintenance [15]. Thus, Norrin canhave developmental and homeostatic functions beyond
the retina [16].
Nucleotide sequence variants in the NDP gene are associated with several congenital retinal pathologies withor without systemic involvement. To date, 167 mutations in the NDP gene have been registered in theHuman Gene Mutation Database (HGMD) – among theseMarakhonov et al. BMC Medical Genetics 2020, 21(Suppl 1):156

129 are associated with Norrie disease phenotype, 20 withfamilial exudative vitreoretinopathy, and 7 with retinopathy of prematurity. Most pathogenic variants of the NDP
sequence (n = 100) are nonsense or missense mutations,and 80% are associated with Norrie disease [17].
In this study, we identified the pathogenic variant
NM_000266.3(NDP):c.385G > T that introduces a PTC
in amino-acid position 129 of Norrin. Molecular analysis
allowed diagnosing Norrie disease in the patient. Severe
ophthalmic phenotype with marked systemic involvement is believed to be linked to the absence of theultimate cysteine, which forms the dimeric structure ofmature Norrin and is in accordance with previous observations [18]. We suppose that in the case of earlier diagnosis, the family could have had much greater
opportunity to give a healthy birth avoiding lifethreatening complications, as well as averting the psychologic tragedy in the family.
Clinical polymorphism and variable expressivity ofhereditary ophthalmic pathology severely complicatesthe establishment of an exact diagnosis and make diagnosis time- and cost-consuming. NGS appears to be themethod-of-choice in complicated cases, which could
substantially hasten the establishment of a diagnosis andgenetic risk estimation. This is much more important incase of extremely rare diseases that are not well-known
to physicians and ophthalmologists.
Abbreviations
ACMG: American College of Medical Genetics and Genomics; HGMD: Human
Gene Mutation Database; mmHg: millimetre of mercury; NGS: Nextgeneration sequencing; OD (oculus dextrus): The right eye; OMIM: Online
Mendelian Inheritance in Man; OS (oculus sinister): The left eye;
PTC: Premature termination codon; RCMG: Research Centre for MedicalGenetics; WES: Whole exome sequencing
Acknowledgements
We thank Dr. Richard H. Lozier for interest in our work and useful assistance.
About this supplement
This article has been published as part of BMC Medical Genetics Volume 21
Supplement 1, 2020: Selected Topics in “Systems Biology and Bioinformatics” 2019: medical genetics. The full contents of the supplement are available online
at https://bmcmedgenet.biomedcentral.com/articles/supplements/volume-21supplement-1.
Authors’ contributions
AVM analyzed and interpreted the patient’s NGS data, performed molecular
genetic experiments, analyzed and interpreted the patient data, and wrote
the manuscript. VVK and MFS performed ophthalmic examination. SAR andIAM performed clinical examination and genetic counseling. TAV contributed
to the analysis of the patient data and preparation of the manuscript. OASand NNV performed prenatal diagnosis. SIK and RAZ designed the study andhelped supervise the project. All authors read and approved the finalmanuscript.
FundingPublication of this article was funded by the Russian Foundation for Basic
Research (RFBR grant № 19-015-00122) and the state assignment of the Ministry of Science and Higher Education of the Russian Federation. The funders
had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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Availability of data and materials
The datasets used and/or analyzed during the current study are available
from the corresponding author on reasonable request.
Ethics approval and consent to participate
The clinical and molecular genetic study was performed in accordance with
the Declaration of Helsinki and was approved by the Institutional Review
Board of the Federal State Budgetary Institution “Research Centre for MedicalGenetics,” Moscow, Russia, with written informed consent obtained fromeach participant and/or their legal representative as appropriate.
Consent for publication
Consent for publication was obtained from the legal guardian of the patient.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Laboratory of Genetic Epidemiology, Research Centre for Medical Genetics,Moscow, Russian Federation. 2S. Fyodorov Eye Microsurgery Federal State
Institution, Moscow, Russian Federation. 3N.A. Semashko National ResearchInstitute of Public Health, Moscow, Russian Federation.
Published: 22 October 2020
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